EP1416276B1 - Biosensors and measurement method - Google Patents

Biosensors and measurement method Download PDF

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EP1416276B1
EP1416276B1 EP20020762765 EP02762765A EP1416276B1 EP 1416276 B1 EP1416276 B1 EP 1416276B1 EP 20020762765 EP20020762765 EP 20020762765 EP 02762765 A EP02762765 A EP 02762765A EP 1416276 B1 EP1416276 B1 EP 1416276B1
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reagent
sample solution
biosensor
reagent immobilization
marker
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German (de)
French (fr)
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EP1416276A1 (en
EP1416276A4 (en
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Masataka Nadaoka
Mie Takahashi
Hirotaka Tanaka
Fumihisa Kitawaki
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Panasonic Corp
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Panasonic Corp
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Priority to PCT/JP2002/008163 priority patent/WO2003014740A1/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

Abstract

A biosensor provided with a development layer for developing a solution sample, and having a reagent part which is immobilized on a part of the development layer and another reagent part which is held in a labeled and dry state on a part of the development layer and can be eluted by developing the solution sample. By this biosensor, a component to be measured in the solution sample is qualitatively or quantitatively measured by quantifying the bonded labeling reagent in the reagent immobilization part. In this biosensor, a prozone phenomenon can be detected by using a plural number of the reagent immobilization parts and varying the affinities of the respective reagent immobilization parts for the subject to be measured in the solution sample or the labeling reagent. Also, a biosensor having a high measurement accuracy whereby the concentration of the subject to be measured in the solution sample can be measured over a broadened dynamic range is provided. Thus, it is possible to provide a biosensor by which a prozone phenomenon can be detected even in case of measuring a subject concentration over a wide range and which has a wide dynamic measurement range, a high precision and a high accuracy.

Description

    TECHNICAL FIELD
  • The present invention relates to a biosensor and a measurement method and, more particularly, to a biosensor utilizing chromatography and a measurement method using the biosensor.
  • BACKGROUND ART
  • Conventionally, there is an immunochromatography sensor as a typical example of a biosensor which is provided with a developing layer for developing a sample solution, includes a reagent part immobilized to a portion of the developing layer and a marked reagent part that is held by a portion of the developing layer in a dry state and is dissolvable by developing the sample solution, and measures the amount of the marker reagent bound to the reagent immobilization part, thereby to qualitatively or quantitatively analyze an analyte in the sample solution.
  • A general example of an immunochromatography sensor is provided with a sample applying part to which a sample solution is applied, and plural developing layers, and an antibody is immobilized to portions of the developing layers. Further, a marker antibody is held at the upper stream than the antibody immobilization part in a dry state so that it is dissolvable by the sample solution. When a required amount of sample solution is applied to the sample applying part, the sample solution penetrates through the developing layers, whereby measurement is started. A result of measurement is detected by the marker antibody that is bound to the antibody immobilization part. Particles of gold colloid are commonly used as a marker, and the binding to the antibody immobilization part is visually observable due to the particles of gold colloid. Thus, the result of measurement is obtained by visual observation. While sandwich reaction of antigen-antibody reaction is employed as a measurement principle, even when competition reaction is employed as a measurement principle, a result of measurement can be obtained by observing the state of binding of the marker reagent to the antibody immobilization part. In this specification, "immune chromatography" and "immunochromatography" denote the same chromatography, and it is an immunomeasurement method in which complexes of an immobilized reagent and a marker reagent are produced in a reaction layer comprising a wettable porous material, thereby to measure an analyte. That is, it is a measurement system utilizing antigen-antibody reaction. While the conventional immunomeasurement method needs a cleaning operation such as B/F separation, in the immunochromatography method, B/F separation is executed during the process in which the sample solution penetrates through a chromatography carrier as a reaction layer. Usually all reagents are in their dry states, and they are wetted by the sample solution during measurement. While gold colloid and latex are common as markers, magnetic particles, enzymes, and metal colloids other than gold colloid may be used. When the marker is an enzyme or the like, a user operation of adding an enzyme substrate or a reaction stopping agent is included as a measurement operation. Further, amongst the above-mentioned immunochromatography methods, one-step immunochromatography is a measurement method in which measurement is carried out by only a user operation of adding a sample solution. Since the fundamental measurement operation by the user is only application of a sample solution, it is called one-step immunochromatography. Further, although the above-described method requires qualitative judgement by visual observation, when a desired result of measurement is semi-quantitative or when judgement with accuracy higher than that is required, there is employed a method of reading a result of measurement by a transparent mode using an optical reading device, which is disclosed in Japanese Published Patent Application No. Hei. 10-274624, or a method of capturing a result of measurement as an image with a camera or the like, and arithmetically processing the image, which is disclosed in Japanese Published Patent Application No. Hei.10-274653.
  • On the other hand, examples of a sensor device having the function of performing quantitative analyze by itself without requiring a measurement device for directly detecting a signal from the sensor by visual observation, have been disclosed in Japanese Patent No.3005303, Japanese Published Patent Application No. Hei.7-159398, Japanese Published Patent Application No. Hei.8-278305. These inventions provide a sensor having the function of quantitative analysis by detecting the number of parts to which a marker reagent is bound among plural reagent immobilization parts, a sensor having the function of semi-quantitative analysis by varying the concentration in a reagent immobilization part, and a sensor which can simultaneously measure different target items in plural reagent immobilization areas.
  • In recent years, POCT (Point-of-Care Tests) is gradually becoming widespread in medical diagnosis scenes. In POCT, especially, a device that can measure an analyte speedily, easily, and precisely is desired. A fundamental principle employed for POCT has convenience that can deal with a wide range of analytes, and it is progressing in various fields not only clinical fields but also food hygiene fields, environmental measurement fields, and the like. On the other hand, although some POCT have quantitativeness for limited target items, most of POCT have only qualitative or semi-quantitative accuracy, and therefore, a technique that can measure an analyte more speedily, easily, and accurately and is applicable to wider fields has been demanded. However, while in the above-described method the analyte is measured by detecting the amount of the marker reagent bound to the reagent immobilization part in the sensor, the binding of the marker reagent to the reagent immobilization part has limitations. First of all, in the case of using sandwich reaction, a measurable antigen concentration area is eventually limited. Especially when it is antigen-antibody reaction, the antigen concentration in the area where the amount of binding linearly increases is about single or double digits. Even when more target antigen exists, it is saturated at a predetermined amount of binding, and the antigen exceeding the saturation level cannot be bound to the reagent immobilization part. When the target antigen further increases, a prozone phenomenon occurs. Therefore, when the concentration of the target antigen is high, previous dilution is needed. In order to perform dilution as well as execute highly precise quantitative analysis, dilution precision is also needed as a matter of course, and a device for dilution is required and, further, a dilution operation is required. Such dilution operation is extremely complicated for unskilled persons having little experience of chemical experiments, and therefore, the user must be selected. Furthermore, when such operational precision is not required, dilution can be carried out with relative ease by using a common pipette or the like. In this case, however, precision cannot be expected. Moreover, since the dilution operation is needed in addition to the measurement operation, extra time is required. Therefore, when speedy measurement in POCT is required, the measurement method using sandwich reaction can be used for only lower-accuracy qualitative analysis or semi-quantitative analysis. Further, a serious problem of the prozone phenomenon resides in that, even when the concentration of the actual analyte in the sample solution is high, a result apparently equivalent to a low concentration is undesirably obtained. For example, in the case of measurement in a clinical test, since a prescription for a patient is selected according to the test result, such prozone phenomenon might cause, in extreme cases, a problem relating to continuation of life. Accordingly, false-negative (FN) due to prozone phenomenon can be a fatal problem for the measurement.
  • Next, in the case of using competitive reaction, the amount of the marker reagent bound to the reagent immobilization part decreases with an increase in the concentration of the target antigen, and the marker reagent is not bound to the reagent immobilization part when the concentration of the target antigen is higher than a predetermined level. Also in this competitive reaction, when an antibody and an antigen are used as the immobilized reagent components, the target antigen concentration area is eventually limited due to the nature of binding, and a dilution operation is needed when the concentration of the target antigen is high, as in the above-mentioned sandwich reaction. In order to perform dilution as well as highly-precise quantitative analysis, dilution precision is also required as a matter of course, and a device for dilution is required and, furthermore, a dilution operation is required. Such dilution operation is extremely complicated for unskilled persons having little experience of chemical experiments, and therefore, the user must be selected. Furthermore, when such operational precision is not desired, dilution can be carried out with relative ease by using a common pipette or the like. In this case, however, precision cannot be expected. Moreover, since the dilution operation is needed in addition to the measurement operation, extra time is required. Therefore, when speedy measurement in POCT is required, the measurement method using competitive reaction can be used for only lower-accuracy qualitative analysis or semi-quantitative analysis. Further, only analytes having less change in target antigen concentration can be selected. Moreover, in order to measure an analyte having a wide concentration range without performing dilution, plural sensor devices must be used. When plural sensor devices are used, since the concentration of the analyte in the employed sample solution is not known by the operator, the operator must perform measurement twice, resulting in complicated workability and increased costs.
  • DISCLOSURE OF THE INVENTON
  • In order to solve the above-described problems, there is provided a biosensor which has a developing layer for developing a sample solution, includes a reagent part immobilized to a portion of the developing layer and a marked reagent part that is held by a portion of the developing layer under a dry state and is dissolvable by developing the sample solution, and measures the amount of the marker reagent bound to the reagent immobilization portion, thereby to qualitatively or quantitatively analyze an analyte in the sample solution; wherein plural reagent immobilization parts exist, and the plural reagent immobilization parts have different affinities for the analyte in the sample solution or the marker reagent. Since the plural reagent immobilization parts are provided and the respective parts have different affinities for the analyte in the sample solution or the marker reagent, a prozone phenomenon can be detected, and furthermore, the dynamic range of the concentration of the analyte in the sample solution can be increased. The "prozone phenomenon" described in this specification indicates, for example, an antigen excess area and a post-zone area in measuring an antigen in an antigen-antibody reaction. When a sandwich reaction system in the above-described immunochromatography sensor is taken as an example, complexes of immobilized reagent, analyte, and marker reagent are generated in the reagent immobilization parts in accordance with the concentration of the analyte in the sample solution, and the amount of the complexes to be formed increases as the concentration of the analyte in the sample solution increases. However, when the concentration of the analyte reaches a predetermined level, the complex formation amount is saturated. When the concentration of the analyte exceeds the level, the complex formation amount decreases. At last, the concentration of the analyte reaches an area where no complex is formed. The phenomenon that occurs in the area where the complex formation amount decreases and the area where no complex is formed at all although the analyte exists at a high concentration, is called "prozone phenomenon". While the prozone phenomenon is described with respect to the sandwich reaction in the immunochromatography sensor having an antigen as an analyte, it is needless to say that this phenomenon also occurs when the analyte is an antibody in a sandwich reaction system which forms complexes in similar manner, or in a reaction system utilizing a binding reaction. Further, the above-described analyte dynamic range means the measurable range of the concentration of the analyte in the test solution. For example, depending on the measurement method, there are cases where the concentration of the original sample solution is measured as it is, or where the measurable range is made wider by dilution or the like. However, the dynamic range described here is a pure measurable range in the case where the sample solution is used as it is, without adding a diluent or the like. The dynamic range will be described taking a perfect dry system immunochromatography sensor as an example. At present, there is an immunology test for pregnancy using urine as a specimen, which is commonly used in clinical scenes or homes. In this case, the user drops urine onto a sensor device to complete an operation relating to measurement, and checking a test result is only left for the user to do. That is, in this case, the range of concentration that is actually measurable when urine is dropped as it is, is called an analyte dynamic range. This is merely an example, and the same can be said of other analytes, samples, and reaction modes.
  • The present invention is made to solve the above-described problems and has for its object to detect a prozone phenomenon by making the plural reagent immobilization parts have different affinities for the analyte or the marker reagent even when the concentration of the analyte in the sample solution is high. Furthermore, it is another object of the present invention to provide a biosensor which can measure a wider range of concentration of the analyte by making the plural reagent immobilization parts have different affinities, and therefore, can select analytes over a wide range.
  • According to Claim 1 of the present invention, there is provided a biosensor having a developing layer for developing a sample solution, including a reagent part immobilized to a portion of the developing layer and a marked reagent part which is held in a dry state by a portion of the developing layer, and is dissolvable by developing the sample solution, and qualitatively or quantitatively analyzing an analyte in the sample solution by measuring the amount of the marker reagent bound to the reagent immobilization part; wherein plural reagent immobilization parts exist, and the respective reagent immobilization parts have different affinities for the analyte in the sample solution or the marker reagent. The biosensor is characterized by that plural reagent immobilization parts are provided, and the respective reagents have different affinities for the analyte in the sample solution or the marker reagent.
  • According to Claim 2 of the present invention, there is provided a biosensor which is a device having a developing layer for developing a sample solution, including a reagent part immobilized to a portion of the developing layer and a marked reagent part which is held in a dry state by a portion of the developing layer, and is dissolvable by developing the sample solution, and having a sample applying part on which the sample solution is applied, the marker reagent part, and the marker immobilization part which are arranged in this order, said biosensor qualitatively or quantitatively analyzing an analyte in the sample solution by measuring the amount of the marker reagent bound to the reagent immobilization part; wherein plural reagent immobilization parts exist, and the respective reagent immobilization parts have different affinities for the analyte in the sample solution or the marker reagent. The biosensor is a device having the sample applying part, the marker reagent part, and the reagent immobilization parts in this order, and further, it is characterized by that plural reagent immobilization parts exist, and the respective reagent immobilization parts have different affinities for the analyte in the sample solution or the marker reagent.
  • According to Claim 3 of the present invention, in the biosensor as defined in Claim 1 or 2, the reagents immobilized to the plural reagent immobilization parts are antibodies, the analyte in the sample solution is an antigen, and an antibody having a higher affinity for the analyte in the sample solution or the marker reagent is immobilized to the reagent immobilization part that is positioned on the upper stream side with respect to the sample solution applying part. In the biosensor as defined in Claim 1 or 2, the reagent immobilization parts are antibodies, and the analyte in the sample solution is an antigen, and further, an antibody having a higher affinity for the antigen is provided on a part at the upper stream side in the sample penetrating and developing direction with respect to the sample solution applying part, that is, the antibody is provided on a part which earlier comes in contact with a developing mixture solution which develops while dissolving the marker material after the sample solution is applied to start development.
  • According to Claim 4 of the present invention, in the biosensor as defined in any of Claims 1 to 3, the reagents in the plural reagent immobilization parts are monoclonal antibodies. In the biosensor as defined in any of Claims 1 to 3, each of the reagents on the plural reagent immobilization parts is a monoclonal antibody.
  • According to Claim 5 of the present invention, in the biosensor as defined in any of Claims 1 to 4, the analyte in the sample solution is quantitatively analyzed by measuring the amount of the marker reagent bound to the plural reagent immobilization parts. In the biosensor as defined in any of Claims 1 to 4, the analyte in the sample solution is measured by measuring the amount of the marker reagent bound to the reagent immobilization parts.
  • According to Claim 6 of the present invention, in the biosensor as defined in any of Claims 1 to 5, a prozone phenomenon is detected by measuring the amount of the marker reagent bound to the plural reagent immobilization parts. In the biosensor as defined in any of Claims 1 to 5, by measuring the marker reagent binding states in the plural reagent immobilization parts, it is detected whether or not the respective parts are prozone areas in the measurement. Although the prozone area has already been described, the prozone area described in this specification indicates, for example, an antigen excess area and a post zone area in measuring an antigen in an antigen-antibody reaction. When a sandwich reaction system in the immunochromatography sensor is taken as an example, complexes of immobilized reagent, analyte, and marker reagent are generated in the reagent immobilization parts in accordance with the concentration of the analyte in the sample solution, and the amount of the complexes to be formed increases as the concentration of the analyte in the sample solution increases. However, when the concentration of the analyte reaches a predetermined level, the complex formation amount is saturated. When the concentration of the analyte is higher than the level, the complex formation amount decreases. When the concentration of the analyte further increases, it reaches an area where no complex is formed. A part which is generally called a zone area or a zone phenomenon, including the area where the complex formation amount decreases and the area where no complex is formed at all although the analyte exists at a high concentration, is called a prozone area. While the sandwich reaction in the immunochromatography sensor is taken as an example, it is needless to say that a prozone area is a phenomenon that also occurs when the analyte is an antibody in a sandwich reaction system which forms complexes in similar manner, or in a reaction system utilizing binding reaction.
  • According to Claim 7 of the present invention, in the biosensor as defined in any of Claims 1 to 6, among the plural reagent immobilization parts, the amount of the marker reagent bound to the reagent immobilization part which is positioned on the uppermost stream side with respect to the sample solution applying part is measured, thereby to measure the analyte in the sample solution; and the amounts of the marker reagent bound to the other reagent immobilization parts are also measured and, on the basis of the results of the respective measurements, the measured value of the amount of the marker reagent bound to the uppermost-steam side reagent immobilization part is subjected to prozone judgement. In the biosensor as defined in any of Claims 1 to 6, the analyte in the sample solution is measured by measuring the amounts of the marker reagent bound to the plural reagent immobilization parts. At this time, in measuring the sample solution, the measurement is carried out using the reagent immobilization part positioned at the uppermost stream side viewed from the sample solution applying part, and the bindings of the marker reagent in the other reagent immobilization parts are subjected to prozone judgement, thereby to judge as to whether the binding of the marker reagent in the uppermost-stream side reagent immobilization part is a prozone area or not.
  • According to Claim 8 of the present invention, in the biosensor as defined in any of Claims 1 to 7, the plural reagent immobilization parts have different affinities for the analyte in the sample solution or the maker reagent, whereby the respective reagent immobilization parts have different dynamic ranges for measuring the concentration of the analyte in the sample solution. In the biosensor as defined in any of Claims 1 to 7, the plural reagent immobilization parts have different affinities for the analyte in the sample solution or the maker reagent, whereby the respective reagent immobilization parts have different dynamic ranges for measuring the concentration of the analyte in the sample solution. The dynamic range means, as already described above, a pure measurable concentration range of the analyte in the case where the sample solution is used as it is, without adding a diluent or the like. The dynamic range will be described taking a perfect dry system immunochromatography sensor as an example. At present, there is an immunology test for pregnancy using urine as a specimen, which is commonly used in clinical scenes or homes. In this case, the user drops urine onto a sensor device to complete an operation relating to measurement, and checking a test result is only left for the user to do. That is, in this case, the actually measurable range when urine is dropped as it is, is called an analyte dynamic range. This is merely an example, and the same can be said of other analytes, samples, and reaction modes. Further, even when an operation such as dilution is required in the measurement system, a detection sensitivity area for the same sample solution and the same analyte is defined as a dynamic range.
  • According to Claim 9 of the present invention, in the biosensor as defined in Claim 8, the plural reagent immobilization parts have different affinities for the analyte in the sample solution or the marker reagent, thereby to increase the dynamic range for measuring the concentration of the analyte in the sample solution. In the biosensor as defined in Claim 8, when the analyte in the sample solution is measured by measuring the amounts of the marker reagent bound to the plural reagent immobilization parts, since the plural reagent immobilization parts have different affinities for the analyte in the same solution or the marker reagent, the respective parts show different responses to the concentration of the analyte in the sample solution, whereby the analyte dynamic range of the sensor device is increased.
  • According to Claim 10 of the present invention, in the biosensor as defined in any of Claims 1 to 9, the plural reagent immobilization parts recognize the same epitope. In the biosensor as defined in any of Claims 1 to 9, the reagents in the plural reagent immobilization parts recognize the same epitope although they have different affinities for the analyte in the sample solution or the marker reagent. Recognizing the same epitope means that the plural reagent immobilization parts are bound to the same binding site although they have different affinities for the binding site.
  • According to Claim 11 of the present invention, in the biosensor as defined in any of Claims 1 to 10, the reagent immobilization parts are provided in two positions. In the biosensor as defined in any of Claims 1 to 10, the plural reagent immobilization parts are provided in two positions.
  • According to Claim 12 of the present invention, in the biosensor as defined in any of Claims 1 to 11, the plural reagent immobilization parts are in contact with each other. In the biosensor as defined in any of Claims 1 to 11, the respective reagent immobilization parts are in contact with each other.
  • According to Claim 13 of the present invention, in the biosensor as defined in any of Claims 1 to 12, the developing layer employs a lateral flow system, the plural reagent immobilization parts are immobilized in lines along a direction perpendicular to the sample solution developing direction the line width is 0.5mm~2.0mm, and the intervals between the lines of the plural reagent immobilization parts are 1.0mm or longer. In the biosensor as defined in any of Claims 1 to 12, the developing layer employs a lateral flow system, the plural reagent immobilization parts are immobilized in lines along a direction perpendicular to the sample solution developing direction, the line width is 0.5mm~2.0mm, and the intervals between the lines of the respective reagent immobilization parts are 1.0mm or longer.
  • According to Claim 14 of the present invention, in the biosensor as defined in any of Claims 1 to 13, all of the reagents including the marker reagent and the immobilized reagents are in their dry states. In the biosensor as defined in any of Claims 1 to 13, all of the reagents including the marker reagent and the immobilized reagents are in dry states. The dry state means the state before measurement is carried out, that is, the state before the reagents are wetted by the sample solution.
  • According to Claim 15 of the present invention, in the biosensor as defined in any of Claims 1 to 14, the sample solution is urine, saliva, or blood. In the biosensor as defined in any of Claims 1 to 14, the sample solution is urine, saliva, or blood. The blood includes whole blood containing a material component such as blood corpuscle, blood serum excluding a material component, and blood plasma.
  • According to Claim 16 of the present invention, in the biosensor as defined in any of Claims 1 to 15, the biosensor is used immunochromatography. The biosensor as defined in any of Claims 1 to 15 is used immunochromatography.
  • According to Claim 17 of the present invention, there is provided a measurement method employing a biosensor as defined in any of Claims 1 to 16, wherein the amounts of the marker reagent bound to the plural reagent immobilization parts are measured, thereby to qualitatively or quantitatively analyze the analyte in the sample solution. In the measurement method using a biosensor as defined in any of Claims 1 to 16, the measurement is carried out on the basis of the bindings of the marker reagent to the plural reagent immobilization parts.
  • According to Claim 18 of the present invention, there is provided a measurement method employing a biosensor having a developing layer for developing a sample solution, and including plural reagent parts which are immobilized to portions of the developing layer, and have different affinities for an analyte in the sample solution or a marker reagent, and a reagent part which is marked and held by a portion of the developing layer, and is dissolvable by developing the sample solution; wherein the amounts of the marker reagent bound to the plural reagent immobilization parts are measured, thereby to qualitatively or quantitatively analyze the analyte in the sample solution. In the measurement method, the amounts of the marker reagent bound to the plural reagent immobilization parts are measured to qualitatively or quantitatively analyze the analyte in the sample solution, by employing a biosensor having a developing layer for developing a sample solution, and including plural reagent parts which are immobilized to portions of the developing layer, and have different affinities for an analyte in the sample solution or a marker reagent, and a reagent part which is marked and held by a portion of the developing layer, and is dissolvable by developing the sample solution. The qualitative analysis means two-step judgement represented by positive/negative judgement, and the quantitative analysis includes conversion into numerals, and semi-quantitative analysis having three or more steps.
  • According to Claim 19 of the present invention, in the measurement method as defined in Claim 17 or 18, the method for measuring the amounts of the marker reagent bound to the plural reagent immobilization parts employs an electromagnetic wave. In the measurement method as defined in Claim 17 or 18, an electromagnetic wave is employed in the method for measuring the amounts of the marker reagent bound to the plural reagent immobilization parts.
  • According to Claim 20 of the present invention, in the measurement method as defined in Claim 17 or 19, the method for measuring the amounts of the marker reagent bound to the plural reagent immobilization parts is to measure a diffused electromagnetic wave which is obtained when an electromagnetic wave is reflected. In the measurement method as defined in Claim 17 or 19, the method for measuring the amounts of the marker reagent bound to the plural reagent immobilization parts is to measure a diffused electromagnetic wave which is obtained when an applied electromagnetic wave is reflected.
  • According to Claim 21 of the present invention, in the measurement method as defined in any of Claims 17 to 20, an electromagnetic wave source used for the measurement is scanned with respect to the biosensor, or the biosensor is scanned with respect to the electromagnetic wave source, thereby to measure the amounts of the marker reagent bound to the reagent immobilization parts. In the measurement method as defined in any of Claims 17 to 20, when detecting the amounts of the marker reagent bound to the plural reagent immobilization parts, the electromagnetic wave source is scanned, or the biosensor is scanned.
  • According to Claim 22 of the present invention, the measurement method using a biosensor as defined in any of Claims 17 to 21 is reflection absorbance measurement, wherein a light source is shaped in a line according to the plural reagent immobilization parts being shaped in lines, and the line width of the light source is 1.0mm or shorter. In the measurement method defined in any of Claims 17 to 20, the method for detecting the amounts of the marker reagent bound to the plural reagent immobilization parts is to measure reflection absorbance. In this case, the electromagnetic wave is light, preferably, visible light, and the method for detecting the amounts of the marker reagent bound to the plural reagent immobilization parts is to measure diffused light which is obtained when applied visible light is reflected.
  • According to Claim 23 of the present invention, in the measurement method as defined in any of Claims 17 to 22, the amounts of the marker reagent bound to the plural reagent immobilization parts are respectively measured, thereby to perform prozone judgement. In the measurement method as defined in any of Claims 17 to 22, after the amounts of the marker reagent bound to the plural reagent immobilization parts are respectively measured, a prozone area is judged from one or plural results of measurements.
  • According to Claim 24 of the present invention, in the measurement method as defined in any of Claims 17 to 23, among the plural reagent immobilization parts, the amount of the marker reagent bound to the reagent immobilization part which is positioned on the uppermost stream side with respect to the sample solution applying part is measured; the amounts of the marker reagent bound to the other reagent immobilization parts are also measured; and, on the basis of the results of the respective measurements, the measured value of the amount of the marker reagent bound to the uppermost-stream side reagent immobilization part is subjected to prozone judgement. In the measurement method as defined in any of Claims 17 to 23, among the plural reagent immobilization parts, the amount of the marker reagent bound to the reagent immobilization part which is positioned on the uppermost stream side viewed from the sample solution applying part is measured as the analyte in the sample solution, and it is judged whether the result of measurement in the uppermost-stream side part is a prozone area or not, on the basis of the amounts of the marker reagent bound to the other reagent immobilization parts.
  • According to Claim 25 of the present invention, in the measurement method as defined in any of Claims 17 to 24, among the plural reagent immobilization parts, the amount of the marker reagent bound to the reagent immobilization part that is positioned on the uppermost stream side with respect to the sample solution applying part is measured; the amounts of the marker reagent bound to the other reagent immobilization parts are also measured; it is judged by performing arithmetic processing as to whether each of the measurement results is within a marker reagent binding amount measurement range in the uppermost-stream side reagent immobilization part or within a marker reagent binding amount measurement range in another reagent immobilization part; and one of the marker reagent binding amounts is used as a measurement result. In the measurement method as defined in any of Claims 17 to 24, the analyte in the sample solution is measured by detecting the amounts of the marker reagent bound to the plural reagent immobilization parts are detected. Furthermore, when the analyte in the sample solution is measured on the basis of the amount of the marker reagent bound to the reagent immobilization part that is positioned on the uppermost-stream side viewed from the sample solution applying part, the amounts of the marker reagent bound to the other reagent immobilization parts are also measured, and it is judged by performing arithmetic processing as to which one of the marker reagent binding amounts obtained in the plural reagent immobilization parts, including the uppermost-stream side part, should be used for measurement of the concentration of the analyte in the sample solution, on the basis of the marker reagent binding amounts obtained in the respective reagent immobilization parts including the uppermost-stream side part, and then the analyte in the sample solution is measured on the basis of the reagent binding amount obtained in one of the reagent immobilization parts.
  • According to Claim 26 of the present invention, in the measurement method as defined in any of Claims 17 to 25, the measurement is one-step immunochromatography which is started by the sample solution applying operation. The measurement method defined in any of Claims 17 to 25 is carried out using a biosensor which is a one-step immunochromatography that starts measurement by the sample solution applying operation.
  • According to Claim 27 of the present invention, in the biosensor as defined in any of Claims 1 to 10 and 12 to 16, the reagent immobilization parts are provided in three positions. In the biosensor as defined in any of Claims 1 to 10 and 12 to 16, the reagent immobilization parts are provided in three positions.
  • According to Claim 28 of the present invention, in the biosensor as defined in Claim 27, the reagent immobilization part which is positioned at the uppermost stream side with respect to the sample solution applying part has the highest affinity for the analyte in the sample solution or the marker reagent, and the second and third reagent immobilization parts have the same affinity. In the biosensor as defined in Claim 27, the reagent immobilization part which is positioned at the uppermost stream side with respect to the sample solution applying part has the highest affinity for the analyte in the sample solution or the marker reagent, and the second and third reagent immobilization parts have the same affinity.
  • According to Claim 29 of the present invention, in the measurement method as defined in any of Claims 17 to 26, the reagent immobilization parts are provided in three positions. In the measurement method as defined in any of Claims 17 to 26, the reagent immobilization parts are provided in three positions.
  • According to Claim 30 of the present invention, in the measurement method employing a biosensor as defined in Claim 28, the amounts of the marker reagent bound to the plural reagent immobilization parts are measured, thereby to qualitatively or quantitatively analyze the analyte in the sample solution. In the measurement method employing a biosensor as defined in Claim 28, the amounts of the marker reagent bound to the reagent immobilization parts are measured, thereby to qualitatively or quantitatively analyze the analyte in the sample solution.
  • According to Claim 31 of the present invention, in the measurement method as defined in Claim 30, a prozone area is detected on the basis of the amounts of the marker reagent bound to the two reagent immobilization parts which are positioned at lower stream side with respect to the sample solution applying part, among the three reagent immobilization parts. In the measurement method as defined in Claim 30, a prozone area is detected on the basis of the amounts of the marker reagent bound to the two reagent immobilization parts which are positioned at the lower stream side with respect to the sample solution applying part, among the three reagent immobilization parts.
  • According to Claim 1, there is provided a biosensor having a developing layer for developing a sample solution, including a reagent part immobilized to a portion of the developing layer and a marked reagent part which is held in a dry state by a portion of the developing layer, and is dissolvable by developing the sample solution, and qualitatively or quantitatively analyzing an analyte in the sample solution by measuring the amount of the marker reagent bound to the reagent immobilization part; wherein plural reagent immobilization parts exist, and the respective reagent immobilization parts have different affinities for the analyte in the sample solution or the marker reagent. Therefore, in measuring the sample solution, even when the concentration of the analyte in the solution is high, a dilution operation or the like is not needed, whereby a simple and speedy biosensor can be provided. Further, since detection of prozone area is possible, a simple, speedy, and highly precise biosensor can be obtained.
  • According to Claim 2, there is provided a biosensor which is a device having a developing layer for developing a sample solution, including a reagent part immobilized to a portion of the developing layer and a marked reagent part which is held in a dry state by a portion of the developing layer, and is dissolvable by developing the sample solution, and having a sample applying part on which the sample solution is applied, the marker reagent part, and the marker immobilization part which are arranged in this order, said biosensor qualitatively or quantitatively analyzing an analyte in the sample solution by measuring the amount of the marker reagent bound to the reagent immobilization part; wherein plural reagent immobilization parts exist, and the respective reagent immobilization parts have different affinities for the analyte in the sample solution or the marker reagent. Therefore, a biosensor having a wide dynamic range for the concentration of the analyte in the sample solution can be provided. Furthermore, since detection of prozone area is possible, a simple, speedy, highly precise, and highly versatile biosensor can be obtained.
  • According to Claim 3, in the biosensor as defined in Claim 1 or 2, the reagents immobilized to the plural reagent immobilization parts are antibodies, the analyte in the sample solution is an antigen, and an antibody having a higher affinity for the analyte in the sample solution or the marker reagent is immobilized to the reagent immobilization part that is positioned on the upper stream side with respect to the sample solution applying part. In the case of measuring the antigen, since the antibodies having different affinities for the analyte or the marker reagent are immobilized to the plural reagent immobilization parts, the antigen concentration dynamic range can be kept sufficiently wide. Further, assuming that the sample solution applying part is at the uppermost stream, the reagent immobilization part at the upper stream side has the higher affinity for the analyte or the maker reagent, whereby a biosensor with higher accuracy can be provided in the uppermost-stream reagent immobilization part, while a biosensor with higher accuracy and precision which is capable of prozone detection can be provided in the other reagent immobilization part.
  • According to Claim 4, in the biosensor as defined in any of Claims 1 to 3, the reagents in the plural reagent immobilization parts are monoclonal antibodies. Therefore, when biosensors are mass-produced or when plural biosensors having uniform performance are needed, plural or a large quantity of speedy and precise biosensors showing uniform performances can be produced by the uniform properties of the monoclonal antibodies, in combination with high productivity and productive stability.
  • According to Claim 5, in the biosensor as defined in any of Claims 1 to 4, the analyte in the sample solution is quantitatively analyzed by measuring the amount of the marker reagent bound to the plural reagent immobilization parts. The reagents on the plural reagent immobilization parts have different affinities for the analyte or the marker reagent, and the amounts of the marker reagent bound to the respective parts are not checked by fuzzy visual check but the results of measurement are converted into numerals, whereby a simple, speedy, precise, and accurate biosensor can be obtained.
  • According to Claim 6, in the biosensor as defined in any of Claims 1 to 5, a prozone phenomenon is detected by measuring the amount of the marker reagent bound to the plural reagent immobilization parts. Therefore, a biosensor with higher precision, which can judge whether the amount of the marker reagent bound to each reagent immobilization part is within the prozone area or not, is obtained.
  • According to Claim 7, in the biosensor as defined in any of Claims 1 to 6, among the plural reagent immobilization parts, the amount of the marker reagent bound to the reagent immobilization part which is positioned on the uppermost stream side with respect to the sample solution applying part is measured, thereby to measure the analyte in the sample solution; and the amounts of the marker reagent bound to the other reagent immobilization parts are also measured and, on the basis of the results of the respective measurements, the measured value of the amount of the marker reagent bound to the uppermost-steam side reagent immobilization part is subjected to prozone judgement. Therefore, assuming that the sample solution applying part is the uppermost stream, the amount of the marker reagent bound to the reagent immobilization part on the uppermost stream side among the plural reagent immobilization parts is measured, whereby highly accurate quantitative measurement is realized. Further, prozone judgement is carried out in measuring the amounts of the marker reagent bound to the other reagent immobilization parts, whereby a simple, speedy, and accurate biosensor with higher precision can be obtained.
  • According to Claim 8, in the biosensor as defined in any of Claims 1 to 7, the plural reagent immobilization parts have different affinities for the analyte in the sample solution or the maker reagent, whereby the respective reagent immobilization parts have different dynamic ranges for measuring the concentration of the analyte in the sample solution. Therefore, the plural reagent immobilization parts have different sample solution concentration dynamic ranges, whereby a biosensor which can measure plural analyte dynamic ranges can be obtained.
  • According to Claim 9, in the biosensor as defined in Claim 8, the plural reagent immobilization parts have different affinities for the analyte in the sample solution or the marker reagent, thereby to increase the dynamic range for measuring the concentration of the analyte in the sample solution. Therefore, when measuring the amounts of the marker reagent bound to the plural reagent immobilization parts, measurement over a wider range is realized by combining the analyte concentration dynamic ranges of the respective reagent immobilization parts. Thereby, a biosensor, which can measure the analyte concentration over a wide range by onetime measurement without requiring a complicated operation such as dilution, can be obtained.
  • According to Claim 10, in the biosensor as defined in any of Claims 1 to 9, the plural reagent immobilization parts recognize the same epitope. Therefore, even when the reaction mode in each of the plural reagent immobilization parts is any of "marker reagent-immobilized reagent", "marker reagent", and "analyte-immobilized reagent", a stable, simple, precise, and speedy biosensor, in which stereoscopic damage relating to the reaction in molecular level is small, can be obtained.
  • According to Claim 11, in the biosensor as defined in any of Claims 1 to 10, the reagent immobilization parts are provided in two positions. Therefore, the dynamic range for analyte concentration is increased, and a minimum reagent composition that enables prozone detection is realized, whereby a cheaper, speedy, simple, and precise biosensor can be obtained.
  • According to Claim 12, in the biosensor as defined in any of Claims 1 to 11, the plural reagent immobilization parts are in contact with each other. Although the development of the sample solution on the reagent immobilization parts generally becomes non-uniform, the plural reagent immobilization parts are apparently united into one, resulting in a highly accurate biosensor having a wide analyte dynamic range and being able to perform prozone detection, in which penetration of the sample solution that develops the developing layer is kept more uniform.
  • According to Claim 13, in the biosensor as defined in any of Claims 1 to 12, the developing layer employs a lateral flow system, the plural reagent immobilization parts are immobilized in lines along a direction perpendicular to the sample solution developing direction, the line width is 0.5mm~2.0mm, and the intervals between the lines of the plural reagent immobilization parts are 1.0mm or longer. When the sample solution develops the plural reagent immobilization parts on the developing layer, the development is apt to be non-uniform. However, since the line width is 0.5mm~2.0mm, the development can be visually checked while suppressing the adverse effect of non-uniform development. Further, since the intervals between the reagent immobilization parts are 1.0mm or more, the respective parts can be visually distinguished from each other. Therefore, a simpler, speedier, highly accurate and precise biosensor having excellent viewability can be obtained. This is applicable to the above-mentioned biosensor employing an optical measurement device.
  • According to Claim 14, in the biosensor as defined in any of Claims 1 to 13, all of the reagents including the marker reagent and the immobilized reagents are in their dry states. Since the plural reagent immobilization parts have different affinities for the analyte or the marker reagent, a biosensor having a sufficiently wide dynamic range for the analyte concentration, and a function of detecting a prozone area can be obtained. Moreover, since all of the reagents are in their perfect dry states, a biosensor which has excellent shelf life and stability and is easily portable can be obtained.
  • According to Claim 15, in the biosensor as defined in any of Claims 1 to 14, the sample solution is urine, saliva, or blood. Therefore a highly precise, simple, and speedy biosensor can be provided in the field of clinical examination where speedy reaction is desired.
  • According to Claim 16, the biosensor as defined in any of Claims 1 to 15 is used immunochromatography. Therefore, in the immunochromatography which is becoming widespread on the market as a simple immunomeasurement method, a highly-precise biosensor which prevents the user from performing false judgement, and realizes an operation as simple as that of the conventional immunochromatography, can be obtained.
  • According to Claim 17, in the measurement method employing a biosensor as defined in any of Claims 1 to 16, the amounts of the marker reagent bound to the plural reagent immobilization parts are measured, thereby to qualitatively or quantitatively analyze the analyte in the sample solution. Therefore, even when the concentration of the analyte in the sample solution is high, a dilution operation or the like is not needed in measuring the sample solution, whereby a simple and speedy measurement method can be obtained. Furthermore, since detection of prozone areas is possible, a simple, speedy, yet highly precise measurement can be realized.
  • According to Claim 18, there is provided a measurement method employing a biosensor having a developing layer for developing a sample solution, and including plural reagent parts which are immobilized to portions of the developing layer, and have different affinities for an analyte in the sample solution or a marker reagent, and a reagent part which is marked and held by a portion of the developing layer, and is dissolvable by developing the sample solution; wherein the amounts of the marker reagent bound to the plural reagent immobilization parts are measured, thereby to qualitatively or quantitatively analyze the analyte in the sample solution. Therefore, even when the concentration of the analyte in the sample solution is high, a dilution operation or the like is not needed in measuring the